I am stimulating whole blood directly after collection with LPS and IFNg (trying to avoid fickle-paque). Using the above mentioned Ab, when staining the blood at 0 timepoint, the subset look very well defined and proportioned, but when i stain and analyse blood from 24 and 48 hours, monocytes have been greatly reduced and the subsets become hard to distinguish. I am stimulating on a rotator plate so i don't think that they are sticking to the surface of the tube, Does anyone have experience with this?
Probably, this effect occurs due to the phenomenon of Shedding CD14 on monocytes during activation with LPS and IFN-g. See, please, the article http://www.jimmunol.org/content/147/5/1567.short
It`s an old article, but may be useful for you. The detailed explanation of your experiment and stimulation conditions may invoke another ideas.
Probably, this effect occurs due to the phenomenon of Shedding CD14 on monocytes during activation with LPS and IFN-g. See, please, the article http://www.jimmunol.org/content/147/5/1567.short
It`s an old article, but may be useful for you. The detailed explanation of your experiment and stimulation conditions may invoke another ideas.
Hi Nunzio,
Are you sure the problem is because of the treatment? Or other factors like storage/incubation temperature are affecting the blood samples? Do you leave some of your samples LPS/IFN-g untreated as control to run after 24/48 hours? If you don't keep your samples in 4 degree, the cell number and sample quality decrease.
Despite the use of the rotator plate, the adhesion of monocytes to the plastic surface cannot be excluded. You can try by using glass tubes. In addition, as suggested by Farzaneh, the temperature can have a role in increasing adhesion.
I stimulate monocytes from PBMCs routinely with LPS, and my CD14 expression stays the same and I get the same separation with my stain. Forward and side scatter of the monocytes do change however due to their activation.
Not sure if costimulation with IFNg is causing the problem.
Thank you everyone for sharing your experience
Alexey thank you for this useful article.
Antonio/Farzaneh i have unstimulated samples at everytime point and the monocytes are nonetheless reduced and subsets hard to distinguished, in a previous experiment i considered the possibility of monocyte sticking to the tube, so after incubation at 37'C i bathed the whole blood in ice-cold 2mM EDTA for 10 mins at 4'C as i thought the temperature shock will cause sticky monocyte to detach, but the proportions were very similar to the experiments run without this step. How would you suggest to incubate to avoid sticking of monocytes altogether?
Hi Nunzio
I don't know if the monocytes stickiness to plastic surfaces is reversible by changing the temperature, may be no. I still suggest the use of glass tubes
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